JP2010509207A - Method for producing chlorophyll a and chlorin e6 - Google Patents

Abstract

The present invention relates to a method for producing chlorophyll a and chlorin e6. In the present invention, chlorophyll a is extracted using intact chlorella cells themselves that have not been disrupted, chlorin e6 is produced therefrom, and a high content ratio of chlorophyll a is determined by the pretreatment process of chlorella cells adopted in the present invention. From this, chlorin e6 can be produced with high yield. In addition, the method of the present invention is performed by a relatively simple process and is suitable for mass production of chlorin e6.
[Selection] Figure 3c

Description

  The present invention relates to a method for producing chlorophyll a and chlorin e6.

  Chlorin e6 is a photosensitive substance used for photodynamic therapy of cancer.

  US Pat. No. 5,330,741 discloses a method for producing chlorin e6. According to the method of this patent, the biomass is treated 2-3 times with acetone to extract chlorophyll a. The biomass is then filtered or centrifuged to evaporate the extract and treat the acid to remove magnesium ions from the chlorophyll molecules and hydrolyze the phytyl ester groups. At the same time, methyl alcohol is added for esterification, the reaction mass is treated with water, the pheophorbide α derivative is extracted with chloromethylene, the extract is neutralized, washed with water, Evaporate and chromatograph on alumina. Next, methyl pheophorbide α was crystallized from a mixture of chloromethylene and methanol, and the resulting pheophorbide α derivative was reacted with a strong inorganic base in pyridine-diethyl ether-n-propanol in the presence of oxygen, and reacted. Treat the trout with water. The aqueous layer is acidified to pH 4, the unstable chlorin is extracted with chloromethylene, the extract is evaporated, the unstable chlorin is redissolved in tetrahydrofuran, and the solution is evaporated. Is repeated until the absorbance increase at 700 nm stops. The resulting purpurine 18 is dissolved in tetrahydrofuran, esterified with diazomethane, the purpurine 18 methyl ester is mixed with an aqueous lysine solution in chloromethylene in the presence of pyridine, and the mixture is stirred at room temperature for 12 hours. After removing the solvent under high vacuum, the resulting crude product is purified by reverse phase high performance liquid chromatography (HPLC) and the solvent is removed by lyophilisation. PS is dissolved in a phosphate buffer solution to obtain an injection solution for PDT, 0.1N NaOH solution is added, and the pH is adjusted to a physiological value of pH 7.35 using 0.1N HCl. And the solution is filtered through a microporous filter.

  However, the method has low reproducibility, labor (high vacuum, crystallization, use of column chromatography and HPLC, long reaction time with lysine), and highly toxic and flammable reagents (diazomethane, pyridine, methanol, Tetrahydrofuran, diethyl ether) is not suitable for the pharmaceutical or food industry.

  According to the method of Korean Patent Application Publication No. 2004-0025911, Spirulina biomass is treated with acetone until chlorophyll a is completely extracted, and the biomass is filtered or centrifuged. The extract is treated with acid to remove magnesium ions from the chlorophyll molecules, neutralized with the extract, filtered out precipitated pheophytin a, then 6-16 ml acetone, 1 g crude pheophytin, 0 Hydrolyze pheophytin a using a hydrochloric acid-acetone-hexane mixture composed of 6-6 ml hexane and 5-10 ml concentrated hydrochloric acid. The mixture was then stirred for 20 minutes to 1 hour while heating to 40-60 ° C., then hexane (6-16 ml) was added and the organic layer was washed with a mixture of acetone and hydrochloric acid (2-10: 1), After washing the aqueous layer with hexane, pheophorbide a is filtered, washed with water, recrystallized with an acetone-water mixture and dried in air until the weight is constant. Thereafter, pheophorbide a is dissolved in acetone, an aqueous solution containing 0.05 to 1.00% strength inorganic strong base is added, and the mixture is stirred at 30 to 60 ° C. for 5 to 30 minutes. After further addition of the aqueous solution, the mixture was heated at 40-60 ° C. for 20-90 minutes, neutralized with dilute hydrochloric acid, the chlorin e6 precipitate was centrifuged and washed with distilled water until the acid reaction disappeared, 55 ˜80% chlorin e6 is obtained.

  Similarly, the above method has a problem that the process is complicated and a reagent that cannot be applied to food and medicine is used.

  Throughout this specification, numerous patent documents and articles are referenced and their citations are displayed. The disclosures of the cited patent documents and papers are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are explained more clearly.

  In order to develop a method capable of producing chlorin e6 from chlorella belonging to green algae with high yield and capable of producing chlorin e6 by a method suitable for mass production in a relatively simple process. As a result of intensive research, the present invention was completed by developing an efficient manufacturing protocol such as a unique pretreatment process for chlorella.

  Accordingly, an object of the present invention is to provide a method for producing chlorophyll a.

  Another object of the present invention is to provide a method for producing chlorin e6.

  Other objects and advantages of the invention will become more apparent from the detailed description of the invention, the claims and the drawings.

  According to one aspect of the present invention, the present invention provides a method for producing chlorophyll a comprising the step of treating an organic solvent on an intact chlorella that has not been crushed.

  In order to efficiently produce chlorin e6, the present inventors first tried to develop a method capable of producing chlorophyll a efficiently, and for this purpose, chlorella suitable as a starting material was selected.

  According to the conventional method, when extracting chlorophyll a from chlorella, chlorella cells were first disrupted and chlorophyll a was extracted from this lysate. However, the present inventors have deviated from the conventional concept and succeeded in producing chlorophyll a in a very high yield by using intact chlorella that has not been crushed. Such a successful extraction of chlorophyll a cannot be conceived from the prior art, and is a very surprising result.

  As used herein, the term 'intact chlorella' means chlorella that has not been crushed. The term 'intact chlorella' has the same meaning as the term 'live chlorella' commonly used in the art. Usually, when a substance in a cell is extracted or separated, a cell disruption process (for example, ultrasonic grinding) is always accompanied. However, the present invention utilizes chlorella itself that has not been crushed.

  When chlorella cells themselves that are not crushed are used in this way, not only the extraction efficiency of chlorophyll a is high, but also problems such as oxidation of chlorophyll a that occur in the cell crushing process can be prevented.

  Organic solvents that can be used in the method of the present invention include various organic solvents known in the art. For example, (i) C1-C4 anhydrous or hydrous lower alcohol (methanol, ethanol, propanol, butanol, etc.), (ii) Mixed solvent of the lower alcohol and water, (iii) Acetone, (iv) Ethyl acetate , (V) chloroform, (vi) butyl acetate, (vii) 1,3-butylene glycol, (viii) hexane, and (ix) diethyl ether can be used as the organic solvent. Preferably, the organic solvent utilized in the present invention is methanol or ethanol, more preferably ethanol.

  As the chlorella used in the present invention, any ordinary chlorella can be used, and seawater chlorella is preferable. More preferably, the chlorella used in the present invention is Chlorella ellipsoidea, Chlorella minutissima, Chlorella vulgaris, Chlorella fusca, Chlorella zofingiensis, Chlorella stigmataphora, Chlorella vulgaris or Chlorella pyrenoidosa, most preferably Chlorella ellipsoidea.

  According to another aspect of the present invention, the present invention comprises (a) treating chlorella with 30-80% ethanol to remove other components from chlorella other than chlorophyll a; and (b) adding 90% to the chlorella. A method of producing chlorophyll a, comprising: treating -100% ethanol to extract chlorophyll a to obtain a chlorophyll a extract.

  According to yet another aspect of the present invention, the present invention comprises (a) treating chlorella with 80-100% ethanol to extract chlorophyll a; (b) adding dioxane to the extract; And a method for producing chlorophyll a.

  As a result of diligent research to develop a method for efficiently producing chlorophyll a from chlorella as a precursor of chlorin e6, the present inventors have developed two protocols capable of producing chlorophyll a with high yield and purity. did.

Chlorophyll a-Protocol A
Chlorophyll a-Protocol A uses a relatively low concentration of ethanol to remove other components from chlorella than chlorophyll a and uses a relatively high concentration of ethanol to extract chlorophyll a. It has become.

  According to a preferred embodiment of the present invention, the chlorella used in the present invention is an unbroken chlorella cell. In the present invention, it is preferable to carry out using chlorella cells themselves as those that have not been disrupted by sonication or the like. In general, cell lysate is treated with an organic solvent to extract chlorophyll a, but according to a preferred embodiment of the present invention, unbroken chlorella cells themselves are used. When chlorella cells themselves that are not crushed are used in this way, not only the extraction efficiency of chlorophyll a is high, but also problems such as oxidation of chlorophyll a that occur in the cell crushing process can be prevented.

  According to a preferred embodiment of the present invention, the chlorella used in the present invention is a seawater chlorella, more preferably Chlorella ellipsoidea, Chlorella minutissima, Chlorella vulgaris, Chlorella fusca, Chlorella zofingiensis, Chlorella stigmataphora, Chlorella vulgaris or Chlorella pyrenoidosa. And most preferably Chlorella ellipsoidea.

  One of the most significant features of the present invention is that a pretreatment process for removing components other than chlorophyll a from chlorella is performed before extracting chlorophyll a from chlorella. Such a pretreatment process makes it possible to extract chlorophyll a with high purity (content ratio).

  The impurity removal process is performed by treating 30 to 80% ethanol on chlorella. The reason why ethanol is used is that ethanol is the most effective in removing impurities, and secondly, chlorin e6 that is finally produced is expected to be used in food. This is because it is preferable to use ethanol that is harmless to the human body. Ethanol used in the above process has a concentration of 30-80%. If less than 30% ethanol is used, the removal efficiency of impurities is very low. If more than 80% ethanol is used, chlorophyll a is also removed in addition to impurities, and the extraction efficiency of chlorophyll a is greatly reduced. There is.

  Preferably, the concentration of ethanol in step (a) is 30-68%, more preferably 50-65%, even more preferably 58-62%, most preferably about 60%.

  Step (a) is performed at least once, preferably 2-7 times, more preferably 3-6 times, most preferably 5-6 times. When step (a) is repeated, for example, chlorella is suspended in ethanol and centrifuged to remove chlorella impurities by precipitation.

  Chlorophyll a is extracted by treating 90-100% ethanol with chlorella that has undergone a pretreatment process for removing impurities. In this process, if the ethanol concentration is less than 90%, the extraction efficiency is greatly reduced.

  Preferably, the concentration of ethanol in step (b) is 95-100%, more preferably 98-100%, most preferably about 100%.

  The yield with respect to the chlorophyll a of the manufacturing method of the chlorophyll a of the protocol A is 5-15%, Preferably, it is 6-12%, More preferably, it is 7-9%. Yield is calculated as a percentage of the weight ratio of chlorophyll a obtained to 1 g dry weight of chlorella used.

  The purity of chlorophyll-a in protocol A is 70 to 90%, preferably 70 to 85%, more preferably 72 to 80%.

Chlorophyll a-Protocol B
Chlorophyll a-protocol B is a method using high concentrations of ethanol and dioxane. According to this protocol, the process of removing impurities in protocol A described above is omitted, and chlorophyll a can be obtained in a high yield by a simple process. Can be manufactured.

  The contents for chlorella used in chlorophyll a-protocol B are the same as protocol A described above.

  The first stage of chlorophyll a-protocol B is the extraction of chlorophyll a from chlorella using 80-100% ethanol. In this process, if the ethanol concentration is less than 80%, there is a problem that the extraction efficiency is greatly reduced. Preferably, the concentration of ethanol in step (a) is 90-100%, more preferably 98-100%, most preferably about 100%.

  The second stage of the chlorophyll a-protocol B is a stage in which chlorophyll a is precipitated using dioxane. Through this step, not only can chlorophyll a be precipitated to obtain solid phase chlorophyll a, but also the degree of purification of chlorophyll a can be increased. The amount of dioxane used is not particularly limited, but preferably 50 to 250 ml, more preferably 100 to 200 ml, and most preferably 120 to 160 ml is added to 1 L of chlorophyll a extract. In this case, the dioxane used is 100% dioxane.

  According to a preferred embodiment of the present invention, after adding dioxane, additional water is added to the extract. The amount of water used is not particularly limited, but preferably 50 to 250 ml, more preferably 100 to 200 ml, and most preferably 120 to 160 ml is added to 1 L of chlorophyll a extract.

  According to a preferred embodiment of the present invention, after the step (b), the reaction product of the step (b) is subjected to low temperature treatment at -10 ° C. or lower so that the precipitation of chlorophyll a is further improved. More preferably, the low temperature treatment is performed at −15 ° C. to −30 ° C., most preferably −18 ° C. to −25 ° C.

  The yield with respect to chlorophyll a of the manufacturing method of chlorophyll a of protocol B is 3 to 15%, preferably 4 to 10%, more preferably 4 to 6%. The purity of chlorophyll a in Protocol B is 70 to 98%, preferably 75 to 95%, more preferably 80 to 90%.

  According to another aspect of the present invention, (a) treating chlorophyll a obtained by the above-described method with acid and removing magnesium ions from chlorophyll a to obtain pheophytin a; And a step of treating the pheophytin a with a base to obtain chlorin e6.

  The present inventors have studied to develop a method capable of producing chlorin e6 from chlorella belonging to green algae with high yield and producing chlorin e6 by a method suitable for mass production in a relatively simple process. As a result, an efficient manufacturing process was developed.

  The method for producing chlorin e6 of the present invention is a process for producing chlorin e6 using chlorophyll a obtained by the above-described two methods for producing chlorophyll a.

  More specifically, when chlorophyll a produced according to chlorophyll a-protocol A is used as a starting material, the method of the present invention comprises (a) treating chlorella with 30 to 68% ethanol, and from chlorella to chlorophyll a other than chlorophyll a. Removing other components; (b) treating the chlorella with 80-100% ethanol to extract chlorophyll a to obtain a chlorophyll a extract; and (c) adding an acid to the chlorophyll a extract. Treating and removing magnesium ions from chlorophyll a to obtain pheophytin a; and (d) treating pheophytin a with a base to obtain chlorin e6.

  More specifically, when chlorophyll a produced according to chlorophyll a-protocol B is used as a starting material, the method of the present invention includes (a) treating chlorella with 80 to 100% ethanol to extract chlorophyll a. , (B) adding dioxane to the extract to induce precipitation of chlorophyll a, (c) treating the chlorophyll a with acid and removing magnesium ions from chlorophyll a to obtain pheophytin a And (d) treating the pheophytin a with a base to obtain chlorin e6.

  On the other hand, the method for producing chlorin e6 of the present invention basically includes a step of producing pheophytin a using an acid and a step of producing chlorin e6 using a base. More specifically, there are roughly two protocols. Divided.

Chlorine e6-protocol A
Chlorin e6-protocol A is characterized in that chlorin e6 is produced from pheophytin a in an ethanol solvent.

  According to chlorin e6-protocol A, pheophytin a is obtained by treating chlorophyll a extract with acid to remove magnesium ions from chlorophyll a. The chemical formulas of chlorophyll a and pheophytin a are as shown in the following chemical formulas 1 and 2.

  When an acid is treated to chlorophyll a, magnesium ions are removed from Chemical Formula 1 to produce pheophytin a. Preferably, step (a) is performed by treating the chlorophyll a extract with an acid and adjusting the pH of the chlorophyll a extract to 1-5. In this case, if pH 5 is exceeded, there is a problem that the magnesium ion removal rate is greatly reduced. More preferably, the pH of the reactants in step (a) is 1-3, most preferably 2-3.

  A variety of acids known in the art can be used as the acid used in step (a), preferably an inorganic acid, more preferably sulfuric acid and hydrochloric acid, and most preferably hydrochloric acid. is there.

  The pheophytin a produced in the above process is treated with a base to finally obtain chlorin e6 of Formula 3.

  According to a preferred embodiment of the present invention, the step (b) is performed by treating the pheophytin a with a basic solution to adjust the pH to 11 to 16, more preferably 13 to 14. A variety of bases known in the art can be used as the base used in step (b), preferably an inorganic base, and most preferably NaOH.

  According to a preferred embodiment of the present invention, the step (b), ie the step of obtaining chlorin e6, is performed in an ethanol solvent.

  According to a preferred embodiment of the present invention, the reaction product of step (a) is stored in a freezer to induce precipitation.

  Pheophytin a is usually obtained in the form of a precipitate. When chlorin e6 is produced using an ethanol solvent, the pheophytin a precipitate is not directly well dissolved in the ethanol solvent. Therefore, it is preferable to dissolve the pheophytin a precipitate with acetone before the pheophytin a precipitate is dissolved in the ethanol solvent. Next, after removing acetone from the acetone solution (for example, by evaporation), ethanol is added to pheophytin a to dissolve pheophytin a, and then reacted with a base to produce chlorin e6.

  According to a preferred embodiment of the present invention, chlorin e6-protocol A comprises (a) treating chlorophyll a obtained by the above-mentioned method with acid, and removing magnesium ions from chlorophyll a, thereby forming a pheophytin a precipitate. (B-1) adding an acetone solvent to the pheophytin a precipitate to obtain a pheophytin a solution, and then removing the acetone solvent; and (b-2) adding ethanol to the remaining pheophytin a. And (b-3) treating the base with the pheophytin a solution to obtain chlorin e6.

  According to a preferred embodiment of the present invention, after the step (b), the method further includes a step of neutralizing the reaction result of step (b). In this case, neutralization can be performed using various bases, for example, neutralization can be performed using HCl.

After producing chlorin e6, chlorin e6 can be treated with a salt to have a suitable salt form. For example, a salt such as NaHCO ₃ or NH ₄ CO ₃ can be treated to obtain a salt of chlorin e6 in which sodium or ammonium is bound to the —COOH group of chlorin e6.

  The degree of purification of chlorin e6-protocol A relative to chlorin e6 is 80 to 99%, preferably 85 to 99%, and more preferably 93 to 98%. The yield based on chlorin e6 is 3 to 10%, preferably 3 to 8%, more preferably 4 to 6% when chlorella is used as a starting material. Yield is calculated as a percentage of the weight ratio of chlorin e6 obtained to 1 g dry weight of chlorella used.

Chlorine e6-protocol B
Chlorin e6-protocol B is characterized in that chlorin e6 is produced from pheophytin a in an acetone solvent.

  In chlorin e6-protocol B, the explanation for step (a) is the same as chlorin e6-protocol A. In chlorin e6-protocol B, step (b) is basically the same as chlorin e6-protocol A except that pheophytin a is treated with a base in an acetone solvent.

  According to the chlorin e6-protocol B, the pheophytin a precipitate formed in step (a) is dissolved in an acetone solvent, and then the pheophytin a solution is directly treated with a base to produce chlorin e6.

  According to a preferred embodiment of the present invention, chlorin e6-protocol B comprises (a) treating chlorophyll a obtained by the above-described method with an acid and removing magnesium ions from chlorophyll a to obtain a pheophytin a precipitate. And (b-1) adding an acetone solvent to the pheophytin a precipitate to obtain a pheophytin a solution; and (b-2) treating the base with the pheophytin a solution to obtain chlorin e6. Including.

  According to chlorin e6-protocol B, when NaOH is used as a base, chlorin e6 finally produced has a sodium salt form. According to the chlorin e6-protocol A, a separate treatment must be performed to obtain the salt of chlorin e6, but protocol B omits such a process.

  Chlorine e6-protocol B efficiently produces chlorin e6 through a process that is shorter than protocol A. Moreover, although chlorin e6-protocol B consists of the process shortened from protocol A, the purification degree of chlorin e6 finally manufactured is larger than protocol A.

  The purity of chlorin e6-protocol B for chlorin e6 is 90-99.99%, preferably 92-99.95%, more preferably 95-99.90%, most preferably 99.0-99.90%. It is. The yield based on chlorin e6 is 2 to 8%, preferably 3 to 8%, more preferably 3 to 5% when chlorella is used as a starting material.

The features and advantages of the present invention are summarized as follows.
(I) In the present invention, chlorophyll a is extracted using unbroken chlorella cells themselves, and chlorin e6 is produced therefrom.
(Ii) The chlorophyll a having a high content ratio can be obtained by the pretreatment process of the chlorella cells adopted in the present invention, and chlorin e6 can be produced therefrom with a high yield.
(Iii) The method of the present invention is carried out by a relatively simple process and is suitable for mass production of chlorin e6.

FIG. 1a shows the result of extraction for 30 minutes. FIG. 1a is a high pressure liquid chromatography (HPLC) chromatogram showing the result of ethanol extraction of chlorophyll a using pulverized chlorella. The arrow is a peak corresponding to chlorophyll a. It is a HPLC (High pressure liquid chromatography) chromatogram showing the result of ethanol extraction for chlorophyll a using pulverized chlorella. FIG. 1b shows the result of extraction for 1 hour and 30 minutes. The arrow is a peak corresponding to chlorophyll a. It is a HPLC (High pressure liquid chromatography) chromatogram showing the result of ethanol extraction for chlorophyll a using pulverized chlorella. FIG. 1c shows the result of extraction for 3 hours. The arrow is a peak corresponding to chlorophyll a. It is a HPLC chromatogram which shows the result of ethanol extraction with respect to chlorophyll a using the non-pulverized intact chlorella. FIG. 1d shows the result of extraction for 30 minutes. The arrow is a peak corresponding to chlorophyll a It is a HPLC chromatogram which shows the result of ethanol extraction with respect to chlorophyll a using the non-pulverized intact chlorella. FIG. 1e shows the result of extraction for 1 hour and 30 minutes. The arrow is a peak corresponding to chlorophyll a FIG. 1f is an HPLC chromatogram showing the result of ethanol extraction of chlorophyll a using unmilled intact chlorella. FIG. 1f shows the result of extraction for 3 hours. The arrow is a peak corresponding to chlorophyll a It is the HPLC chromatogram which analyzed the component contained in the intact chlorella which is not grind | pulverized. The arrow is a peak corresponding to chlorophyll a. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2b is the result of the first analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2c is the result of the second analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2d shows the results of the third analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2e shows the results of the fourth analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2f shows the results of the fifth analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of having wash | cleaned chlorella with 60% ethanol, and removed other components other than chlorophyll a. FIG. 2g shows the results of the sixth analysis in repeated washing with 60% ethanol. The HPLC analysis is on the supernatant formed as a result of washing. It is a HPLC analysis result which shows the result of extracting chlorophyll a with 100% ethanol. The arrow is a peak corresponding to chlorophyll a. It is a HPLC chromatogram with respect to chlorophyll a manufactured by the manufacturing method of chlorophyll a using a dioxane. The arrow is a peak corresponding to chlorophyll a. 2 is an HPLC chromatogram for pheophytin a formed according to the present invention. The arrow is a peak corresponding to pheophytin a. It is a HPLC chromatogram with respect to chlorin e6 manufactured by the protocol A of this invention. The arrow is a peak corresponding to chlorin e6. It is a HPLC chromatogram with respect to chlorin e6 manufactured by the protocol B of this invention. The arrow is a peak corresponding to chlorin e6.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these examples. It will be obvious to those skilled in the art to which the present invention pertains.

Example 1: Selection of suitable chlorella as starting material As a starting material to finally obtain chlorin e6, ultrasonically crushed chlorella and intact chlorella that was not pulverized [sea chlorella, chlorella ellipsoidea, Chlorland co , Ltd] were prepared. Next, it was confirmed how much chlorophyll a and other components of ultrasonically ground chlorella and intact chlorella were contained. 10 g each of ultrasonic pulverized chlorella and seawater chlorella were taken, 100 ml of ethanol was added and stirred for 30 minutes, 1 hour 30 minutes, 3 hours and 12 hours, and then filtered to obtain an extract solution. The extracted solution was subjected to HPLC analysis, and HPLC was performed using an HPLC system with an SPD-M10AVP column (Dong-il Shimadzu Corp.), the injection volume was 20 μl, and the flow rate was 1 ml / min. As reference, chlorophyll a of Fluka company was used. In FIGS. 1a to 1f, a peak corresponding to a retention time of about 19.5 minutes corresponds to chlorophyll a.

  As can be seen from FIGS. 1a to 1f, when using an intact chlorella, about 20 times as much chlorophyll a can be obtained as compared to using an ultrasonically pulverized chlorella. Therefore, it can be seen that using intact chlorella rather than ultrasonically pulverized chlorella increases chlorophyll a yield and results in more chlorin e6. The present inventors have found the surprising fact that ethanol extraction using chlorella itself is more excellent in extraction efficiency of chlorophyll a than ethanol extraction using a pulverized product obtained by ultrasonically pulverizing chlorella. Such a new discovery is very important because it not only provides the advantage of shortening the chlorophyll purification process but also avoids the problem of oxidization of chlorophyll a during the ultrasonic crushing process.

Example 2: Obtaining Chlorophyll a from Chlorella Protocol A
A-1: Removal of impurities other than chlorophyll a from chlorella First, in order to remove salt from seawater chlorella (Chlorella ellipsoidea, Chlorland co, Ltd), it was washed with distilled water and then centrifuged at 5000 rpm. Precipitated. The precipitate was then resuspended with distilled water and centrifuged again. After performing the washing process with distilled water five times, a small amount of precipitate was taken, and it was confirmed by HPLC how much chlorophyll a and other components were contained. HPLC was performed using an HPLC system with an SPD-M10AVP column (Dong-il Shimadzu Corp.), the injection volume was 20 μl, and the flow rate was 1 ml / min. As reference, chlorophyll a of Fluka company was used. The HPLC results are shown in Figure 2a.

  As can be seen from FIG. 2a, a peak corresponding to chlorophyll a is observed at a retention time of about 9.7 minutes, and peaks corresponding to other various components at a retention time of 2.5 to 7.5 minutes. Was observed.

  Add 100 ml of 60% ethanol to 100 g of chlorella from which seawater has been removed (unbroken chlorella cells themselves), and then centrifuge at 5000 rpm to precipitate chlorella, with a residence time of 2.5 to 7.5 minutes. Some impurities were removed. This ethanol washing process was repeated 6 times. 2b to 2g are HPLC results for the supernatant formed after centrifugation in the ethanol washing process.

  As can be seen from FIGS. 2 b to 2 g, it can be seen that a large amount of impurities appearing at a residence time of 2.5 to 7.5 minutes are removed in the 60% ethanol washing process.

  On the other hand, when the washing process was performed using ethanol having a concentration of 80% or more, chlorophyll a was extracted, and washing, that is, removal of impurities could not be performed. Moreover, although the washing process was performed using ethanol of 20%, 40% and 50% concentration, the removal rate of impurities did not reach 60% ethanol.

  Thus, it can be seen that 60% ethanol is the most preferred cleaning solution. That is, 60% ethanol effectively extracts other components other than chlorophyll a contained in chlorella cells without extracting chlorophyll a from chlorella, and chlorophyll a is relatively high in the washed chlorella. To be included by content.

A-2: Extraction of chlorophyll a from chlorella from which impurities have been removed I
1 L of 100% ethanol was added to 100 g of chlorella from which impurities were removed, and the mixture was stirred for 3 hours to extract chlorophyll a. Chlorella used in the extraction process was not crushed by sonication or the like, and was extracted using the chlorella cells themselves. In general, chlorophyll a is extracted by treating a cell disruption with an organic solvent. In this example, unbroken chlorella cells themselves were used. The extraction result is shown in FIG. 2h.

  As can be seen from FIG. 2h, a peak corresponding to chlorophyll a is observed at a residence time of about 8.4 minutes, and a considerable amount of impurities observed for the residence time of 2.5 to 7.5 minutes in FIG. 2a are removed. I found out. Further, it was confirmed by HPLC chromatogram that the content of chlorophyll a (that is, the degree of purification) was 76.92%. On the other hand, the yield of protocol A based on chlorophyll a was 7.89%.

Protocol B
First, in order to remove salt from seawater chlorella (Chlorella ellipsoidea, Chlorland co, Ltd), it was washed with distilled water and then centrifuged at 5000 rpm to precipitate chlorella. The precipitate was then resuspended with distilled water and centrifuged again. After performing the washing process with distilled water five times, a small amount of precipitate was taken, and it was confirmed by HPLC how much chlorophyll a and other components were contained. HPLC was performed using an HPLC system with an SPD-M10AVP column (Dong-il Shimadzu Corp.), the injection volume was 20 μl, and the flow rate was 1 ml / min. As reference, chlorophyll a of Fluka company was used. 100 ml of ethanol was added to 100 g of chlorella from which seawater had been removed (uncrushed chlorella cells themselves) and centrifuged at 5000 rpm to precipitate chlorella, and only the supernatant chlorophyll extract solution was filtered. After adding 70 ml of dioxane and 70 ml of distilled water to the chlorophyll extract, it was stored in a -20 ° C. freezer for 5 hours or more to induce precipitation. Next, the precipitate was filtered and washed with water to obtain chlorophyll a. The precipitate was then dissolved in 100% ethanol for the following chlorin e6 production.

  As can be confirmed from FIG. 2i, a main peak corresponding to chlorophyll a was observed at a residence time of about 4.3 minutes, and other peaks were hardly generated. It was also confirmed by HPLC chromatogram that the content of chlorophyll a (ie, the degree of purification) was 84.93%. On the other hand, the yield of protocol B with respect to chlorophyll a was 4.26%.

  The experimental results show that when dioxane is used, chlorophyll a can be obtained in a high yield through a shorter process.

Example 3: Production of chlorin e6 from chlorophyll a Example 3-1: Production of pheophytin a 5-10 ml of 1N HCl per liter of ethanol extract containing chlorophyll a obtained from protocol A of Example 2 By processing, the pH of the extract was adjusted to 2-3, and stirred for 3 hours to remove magnesium ions from chlorophyll a to obtain pheophytin a (FIG. 3a). Next, the black pheophytin a solution was stored in a −23 ° C. freezer for 3 to 6 hours to form a precipitate and filtered.

Example 3-2: Production of chlorin e6
Protocol A
After the pheophytin a precipitate of Example 3-1 was dissolved in acetone, the acetone was removed by evaporation. Next, pheophytin a is dissolved in 1 L of 100% ethanol, and 5-10 ml of 1N NaOH is added thereto. The pH of the filtrate containing the pheophytin a solution is adjusted to 13-14 and stirred for 12 hours. Chlorin e6 was formed from a. Thereafter, 1N HCl was added to the reaction product to neutralize it, and after filtration, ethanol was completely removed (FIG. 3b). As can be confirmed from the graph of FIG. 3b, a peak corresponding to chlorin e6 was observed at a residence time of about 4.987 minutes, and the content (purification degree) of chlorin e6 was 97.66%.

  Finally, 48.6 g of chlorin e6 was obtained from 1000 g of chlorella (yield: 4.86%).

In order to produce the salt form of chlorin e6, 3 equivalents of NaHCO ₃ or NH ₄ HCO ₃ was dissolved in ice water, then added to chlorin e6, water was filtered, freeze-dried, Chlorine e6 was produced.

Protocol B
The pheophytin a precipitate of Example 3-1 was dissolved in 1 L of acetone, and then 5-10 ml of 1N NaOH aqueous solution was added to adjust the pH to 13-14, followed by stirring for 12 hours to form chlorin e6 from pheophytin a. I let you. The produced precipitate of chlorin e6 Na salt was filtered, washed with acetone and hexane in order, dissolved in a small amount of water, filtered through a water-soluble membrane filter, and then frozen. Drying gave the final chlorin e6 Na salt (Figure 3c). The final chlorin e6 Na salt showed a powder form. The yield of the final Na salt chlorin e6 is 25-30 g when treated with 10 L of chlorella (1-1.5 kg after centrifugation), and is 2.5-3.0%. The purification degree for chlorin e6 was 99.83%.

  According to Protocol B, the Na salt of chlorin e6 is directly formed by the process of producing chlorin e6 by NaOH treatment. Even in this aspect, there is a great difference from Protocol A.

  As described above in detail, the present invention extracts chlorophyll a using intact chlorella cells themselves that have not been disrupted, produces chlorin e6 therefrom, and pretreats the chlorella cells adopted in the present invention. Depending on the process, a high content ratio of chlorophyll a can be obtained, and chlorin e6 can be produced therefrom with a high yield. In addition, the method of the present invention is performed by a relatively simple process and is suitable for mass production of chlorin e6.

  Although specific portions of the present invention have been described in detail above, such specific descriptions are merely desirable embodiments for those having ordinary knowledge in the art, and the scope of the present invention is not limited thereto. Obviously, it is not limited. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (28)

  A process for producing chlorophyll a comprising the step of treating an intact chlorella with an organic solvent.   The method according to claim 1, wherein the chlorella is a seawater chlorella.   The method of claim 1, wherein the organic solvent is ethanol. (A) treating 30 to 80% ethanol to chlorella to remove components other than chlorophyll a from chlorella;
(B) treating the chlorella with 90-100% ethanol to extract chlorophyll a to obtain a chlorophyll a extract;
A process for producing chlorophyll a. (A) treating chlorella with 80-100% ethanol to extract chlorophyll a;
(B) adding dioxane to the extract to induce precipitation of chlorophyll a;
A process for producing chlorophyll a. (A) treating chlorophyll a obtained by the method according to claim 4 or 5 with an acid to remove magnesium ions from chlorophyll a to obtain pheophytin a;
(B) treating pheophytin a with a base to obtain chlorin e6;
The manufacturing method of chlorin e6 containing this.   The method according to claim 4 or 5, wherein the chlorella is an intact cell that has not been disrupted.   The method according to claim 4, wherein the chlorella is a seawater chlorella.   The method according to claim 4, wherein the ethanol concentration in the step (a) is 50 to 65%.   The method according to claim 9, wherein the concentration of ethanol in step (a) is 58-62%.   The method of claim 4, wherein the step (a) is repeated 3 to 6 times.   The method according to claim 4, wherein the ethanol concentration in the step (b) is 98 to 100%.   The method according to claim 5, wherein the ethanol concentration in the step (a) is 90 to 100%.   The method according to claim 13, wherein the ethanol concentration in the step (a) is 98-100%.   [6] The method according to claim 5, wherein in the step (b), after adding dioxane, water is additionally added to the extract.   The method according to claim 5, wherein, after step (b), the reaction product of step (b) is treated at a low temperature of -10 ° C or lower.   [5] The method according to claim 4, wherein the yield of chlorophyll a is 7-9% and the degree of purification is 72-80%.   The method according to claim 5, wherein the yield of chlorophyll a is 4 to 6% and the degree of purification is 80 to 90%.   The method according to claim 6, wherein the step (a) is performed by treating the chlorophyll a extract with an acid and adjusting the pH of the chlorophyll a extract to 1 to 5.   The method according to claim 19, wherein the pH is 1 to 3.   [7] The method according to claim 6, wherein the step (b) is performed by treating the pheophytin a with a basic solution and adjusting the pH to 11-16.   The method according to claim 21, wherein the pH is 13-14.   The method of claim 6, further comprising neutralizing the reaction product of step (b) after step (b).   The method according to claim 6, wherein the step (b) is performed in an ethanol solvent.   The method according to claim 6, wherein the step (b) is performed in an acetone solvent. The method
(A) treating the chlorophyll a obtained by the method according to claim 4 or 5 with an acid and removing magnesium ions from the chlorophyll a to obtain a pheophytin a precipitate;
(B-1) adding an acetone solvent to the pheophytin a precipitate to obtain a pheophytin a solution;
(B-2) treating the pheophytin a solution with a base to obtain chlorin e6;
26. The method of claim 25, comprising:   [25] The method according to claim 24, wherein the purity of the method with respect to chlorin e6 is 93 to 98% and the yield with respect to chlorin e6 is 4 to 6%.   [26] The method according to claim 25, wherein the purity of chlorin e6 in the method is 95-99.9% and the yield of chlorin e6 is 3-5%.

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